Hyperglycemia is well-recognized and has long-term complications in diabetes mellitus and diabetic nephropathy. In podocytes, the main component of the glomerular barrier, overproduction of reactive oxygen species (ROS) in the presence of high glucose induces dysfunction and increases excretion of albumin in urine. This suggests an impaired antioxidant defense system has a role in the pathogenesis of diabetic nephropathy. We studied expression of NAD(P)H oxidase subunits by Western blotting and immunofluorescence and the activities of the oxidant enzyme, NAD(P)H, and antioxidant enzymes, superoxide dismutase (<em>SOD</em>), glutathione peroxidase (GPx), and catalase (CAT), in mouse podocytes cultured in a high glucose concentration (<em>3</em>0 mM). We found long-term (<em>3</em> and 5 days) exposure of mouse podocytes to high glucose concentrations caused oxidative stress, as evidenced by increased expression of Nox4 and activities of NAD(P)H oxidase (Δ 182%) and <em>SOD</em> (Δ <em>3</em>9%) and decreased activities of GPx (Δ -40%) and CAT (Δ -<em>3</em>5%). These biochemical changes were accompanied by a rise in intracellular ROS production and accumulation of hydrogen peroxide in extracellular space. The role of Nox4 in ROS generation was confirmed with Nox4 siRNA. In conclusion, high glucose concentration affects the oxidant-antioxidant balance in mouse podocytes, resulting in enhanced generation of superoxide anions and its attenuated metabolism. These observations suggest free radicals may play an important role in the pathogenesis of diabetic nephropathy.

CDDO-Me, a synthetic triterpenoid derived from oleanolic acid, is a promising anticancer agent that has shown strong activity against a wide variety of cancer types in vitro and in vivo. We have previously shown that CDDO-Me induces apoptosis in prostate cancer cells irrespective of their hormonal status. To further understand the proapoptotic mechanism of CDDO-Me, we investigated the role of reactive oxygen species (ROS) in mediating the apoptosis inducing activity of CDDO-Me in LNCaP and PC-<em>3</em> prostate cancer cell lines. Here, we show that CDDO-Me induces ROS generation from both nonmitochondrial and mitochondrial sources, which is associated with the induction of apoptosis as characterized by increased annexin V-binding, cleavage of PARP-1 and procaspases-<em>3</em>, -8, -9, loss of mitochondrial membrane potential and release of cytochrome c. In addition, CDDO-Me inhibited cell survival Akt, NF-kappaB and mTOR signaling proteins. The inhibition of ROS generation by N-acetylcysteine (NAC) or by overexpression of antioxidant enzymes glutathione peroxidase (GPx) and superoxide dismutase-1 (<em>SOD</em>-1) prevented CDDO-Me-induced apoptosis. Pretreatment with NAC blocked annexin V-binding, cleavage of PARP-1 and procaspases-<em>3</em>, -8, -9, loss of mitochondrial membrane potential and release of cytochrome c by CDDO-Me. NAC also prevented the inhibition of constitutively active Akt, NF-kappaB and mTOR by CDDO-Me. Together, these data indicate that ROS plays an essential role in the induction of apoptosis by CDDO-Me in prostate cancer cells.

The high event-free survival rates of Down syndrome (DS) children with acute myeloid leukemia (AML) are due, in part, to increased in vitro sensitivity of DS myeloblasts to cytosine arabinoside (ara-C) and daunorubicin and the greater generation of ara-C triphosphate (ara-CTP) from ara-C compared with myeloblasts from non-DS patients (Taub et al, Blood 87:<em>3</em><em>3</em>95, 1996). This study further explores the molecular basis of chemotherapy sensitivity of DS AML patients by examining the expression of chromosome 21-localized genes in myeloblasts from newly diagnosed AML patients. Transcript levels of two chromosome 21-localized genes, cystathionine-beta-synthase (CBS) and superoxide dismutase (<em>SOD</em>), measured by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), were 12.0- and <em>3</em>. 8-fold higher in DS compared with non-DS myeloblasts (P <.0001 and P <.0001, respectively). Conversely, there were no significant increases in transcripts for 2 other chromosome 21-localized genes, carbonyl reductase and the reduced folate carrier. CBS transcript levels correlated with both in vitro ara-C sensitivity measured by the <em>3</em>-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium-bro mid e (MTT) assay (P =.00<em>3</em>) and the generation of (<em>3</em>)H-ara-C triphosphate (ara-CTP) after in vitro incubations with 5 micromol/L (<em>3</em>)H-ara-C (P =.000<em>3</em>). Transcripts of deoxycytidine kinase were 2.6-fold higher in DS compared with non-DS cells and may be a factor in the enhanced metabolism of ara-C in DS cells. There was no significant correlation of <em>SOD</em> transcripts with in vitro ara-C and daunorubicin sensitivities. Increased CBS transcripts could result in elevated CBS activity, which modulates ara-C metabolism by altering reduced folate pools, deoxycytidine triphosphate pools, S-adenosylmethionine levels, and/or methylation of the deoxycytidine kinase gene. The further identification of the molecular mechanisms of chemotherapy sensitivity of DS AML patients may lead to significant improvements in the treatment and cure of AML.

Recently, the treatment of stroke has focused on antioxidant therapies, where oxidative stress is implicated. The preventive and therapeutic potential of plant compounds on ischemic stroke has been intensively studied because many of them contain antioxidant properties. Genistein, one of the active ingredients in soybean, possesses many bioactivities. In this study, we investigated the potential neuroprotective effects of genistein and its possible mechanism of action in a cerebral ischemia mouse model. Mice were pretreated with genistein (2.5, 5, and 10mg/kg) or vehicle orally once daily for 14 consecutive days before transient middle cerebral artery occlusion was performed. Genistein at doses of 2.5-10mg/kg significantly reduced the infarct volume, improved the neurological deficit and prevented cell apoptosis after ischemia. In addition, genistein pretreatment was shown to inhibit the ischemia-induced reactive oxygen species (ROS) production, enhance the activities of antioxidant enzymes superoxide dismutase (<em>SOD</em>) and glutathione peroxidase (GPx), and decrease levels of malondialdehyde (MDA) in stroke mice. Moreover, genistein reversed the mitochondria dysfunction after ischemia, as evidenced by decreasing mitochondria ROS levels, preventing cytochrome C release to the cytoplasm and inhibiting caspase-<em>3</em> activation. Western blotting showed ischemia activated the ROS-dependent nuclear factor-κB (NF-κB) signaling pathway, and genistein suppressed phosphorylation and activation of the NF-κB p65 subunit, as well as the phosphorylation and degradation of the inhibitor protein of κBα (IκBα). Our findings suggested that genistein has a neuroprotective effect in transient focal ischemia, which may involve regulation of mitochondria-dependent apoptosis pathways and suppression of ROS-induced NF-κB activation.

The modifying effect of dietary exposure to a flavonoid, luteolin (LUT) during the azoxymethane (AOM)-induced colon carcinogenesis was investigated in this study. Aberrant crypt foci (ACF), lipid peroxidation (LPO), enzymic and non-enzymic antioxidants and histopathological analysis were performed. Colon carcinogenesis was induced by injecting 15 mg/body kg weight of AOM, intraperitoneally (i.p.), once in a week for <em>3</em> weeks in male Balb/c mice. AOM-induced mice were treated with LUT (1.2mg of LUT/kg body weight/day orally). After the experimental period, frequency of ACF, levels of thiobarbutaric acid reactive substances (TBARS) and hydroxy radical (OH ) were found to be increased, whereas glutathione (GSH), Vitamins C, E and A were decreased in the plasma and colon of AOM-induced mice. However, LUT treatment to AOM-induced mice significantly decreased the incidence of ACF, levels of TBARS and OH with a concordant increase in non-enzymic antioxidants in plasma and colon tissue. The activities of the antioxidant enzymes such as superoxide dismutase (<em>SOD</em>), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) were found to be decreased due to the induction of colon cancer in mouse. LUT treatment ameliorated the activities of these antioxidant enzymes. The histological study revealed a significant increase in the enlarged nuclei and hyperchromatism of cells in AOM-induced mice whereas LUT significantly reduced the signs in the colon. The immunohistochemical expression of MDA-DNA adduct was studied. In AOM-induced group, the expression was increased and treatment with LUT decreased significantly. The present study depicts that LUT can act as an effective chemopreventive agent against colon cancer.

The oxidative reactivities of four tryptophan metabolites in the kynurenine pathway were examined as a potential mechanism for their reported neurotoxicities and carcinogenicities. Neither quinolinic acid, a neurotoxin, nor its monocarboxylic analogue, picolinic acid, auto-oxidized over a wide pH range. However, <em>3</em>-hydroxyanthranilic acid (<em>3</em>-HAT), a carcinogen, readily auto-oxidized and the reaction rate increased exponentially with increasing pH. <em>3</em>-HAT auto-oxidation likely involves two steps: auto-oxidation of <em>3</em>-HAT to the semiquinoneimine (anthranilyl radical) which oxidizes to the quinoneimine, followed by condensation and oxidation reactions to yield a second carcinogen, cinnabarinic acid. <em>3</em>-HAT auto-oxidation to cinnabarinate required molecular oxygen and generated superoxide radicals and H2O2. Superoxide dismutase (<em>SOD</em>) accelerated <em>3</em>-HAT auto-oxidation 4-fold, probably by preventing back reactions between superoxide and either the anthranilyl radical or the quinoneimine formed during the initial step of auto-oxidation. Catalase did not accelerate <em>3</em>-HAT auto-oxidation, but it did prevent destruction of cinnabarinate by H2O2. Interconversion between oxyhemoglobin and methemoglobin occurred during <em>3</em>-HAT auto-oxidation, although neither form of hemoglobin altered rates of <em>3</em>-HAT auto-oxidation. Mn2+, Mn<em>3</em>+ and Fe<em>3</em>+-EDTA did not directly catalyze cinnabarinate formation in the absence of O2, but they did accelerate cinnabarinate formation under aerobic conditions.

Some polyunsaturated fatty acids (PUFAs), if not all, have been shown to have tumoricidal action, but their exact mechanism(s) of action is not clear. In the present study, we observed that n-6 PUFA linoleic acid (LA) inhibited tumor cell growth at high concentrations (above <em>3</em>00 μM); while low concentrations (100-200 μM) promoted proliferation. Analysis of cell mitochondrial membrane potential, reactive oxygen species (ROS) formation, malondialdehyde (MDA) accumulation and superoxide dismutase (<em>SOD</em>) activity suggested that anti-cancer action of LA is due to enhanced ROS generation and decreased cell anti-oxidant capacity that resulted in mitochondrial damage. Of the three cell lines tested, semi-differentiated colorectal cancer cells RKO were most sensitive to the cytotoxic action of LA, followed by undifferentiated colorectal cancer cell line (LOVO) while the normal human umbilical vein endothelial cells (HUVEC) were the most resistant (the degree of sensitivity to LA is as follows: RKO>> LOVO>> HUVEC). LA induced cell death was primed by mitochondrial apoptotic pathway. Pre-incubation of cancer cells with 100 μM LA for 24 hr enhanced sensitivity of differentiated and semi-differentiated cells to the subsequent exposure to LA. The relative resistance of LOVO cells to the cytotoxic action of LA is due to a reduction in the activation of caspase-<em>3</em>. Thus, LA induced cancer cell apoptosis by enhancing cellular oxidant status and inducing mitochondrial dysfunction.

Jasmonic acid (JA) is regarded as endogenous regulator that plays an important role in regulating stress responses, plant growth and development. To investigate the physiological mechanisms of salt stress mitigated by exogenous JA, foliar application of 2mM JA was done to wheat seedlings for <em>3</em>days and then they were subjected to 150mM NaCl. Our results showed that 150mM NaCl treatment significantly decreased plant height, root length, shoot dry weight, root dry weight, the concentration of glutathione (GSH), chlorophyll b (Chl b) and carotenoid (Car), the activities of superoxide dismutase (<em>SOD</em>), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), enhanced the concentration of malondialdehyde (MDA), hydrogen peroxide (H2O2) and the rate of superoxide radical (O2•-) generation in the wheat seedlings when compared with the control. However, treatments with exogenous JA for <em>3</em> days significantly enhanced salt stress tolerance in wheat seedlings by decreasing the concentration of MDA and H2O2, the production rate of O2•- and increasing the transcript levels and activities of <em>SOD</em>, POD, CAT and APX and the contents of GSH, Chl b and Car, which, in turn, enhanced the growth of salt stressed seedlings. These results suggested that JA could effectively protect wheat seedlings from salt stress damage by enhancing activities of antioxidant enzymes and the concentration of antioxidative compounds to quench the excessive reactive oxygen species caused by salt stress and presented a practical implication for wheat cultivation in salt-affected soils.

OBJECTIVE

Accumulating evidence indicates the presence of functional M<em>3</em> subtype of acetylcholine muscarinic receptors (M(<em>3</em>)-mAChR), in addition to the well-recognized M(2)-mAChR, in the heart of various species including man. However, the pathophysiological role of the cardiac M(<em>3</em>)-mAChR remain undefined. This study was designed to explore the possible role of M(<em>3</em>)-mAChR in cytoprotection of myocardial infarction and several related signaling pathways as potential mechanisms.

METHODS

Studies were performed in a rat model of myocardial infarction and in isolated myocytes.

RESULTS

We found that choline relieved myocardial injuries during ischemia or under oxidative stress, which was achieved by correcting hemodynamic impairment, diminishing ventricular arrhythmias and protecting cardiomyocytes from apoptotic death. The beneficial effects of choline were reversed by the M(<em>3</em>)-selective antagonists but not by the M(2)-selective antagonist. Choline/M(<em>3</em>)-mAChR activated several survival signaling molecules (antiapoptotic proteins Bcl-2 and ERKs), increased endogenous antioxidant reserve (SOD), and reduced apoptotic mediators (proapoptotic proteins Fas and p<em>3</em>8 MAPK) and intracellular Ca2+ overload.

CONCLUSIONS

Choline improves cardiac function and reduces ischemic myocardial injuries via stimulating the cardiac M(<em>3</em>)-mAChRs which in turn result in alterations of multiple signaling pathways leading to cytoprotection. The findings suggest M(<em>3</em>)-mAChR as a new target for drug development for improving cardiac function and preventing cardiac injuries during ischemia/reperfusion.

Oxidative stress is a causal factor and key promoter of a variety of cardiovascular diseases associated with apoptotic cell death by causing deregulation of related genes. Though carvedilol, a β-adrenergic blocker, has been shown to produce cytoprotective effects against cardiomyocyte apoptosis, the mechanisms are not fully understood. The present study was designed to investigate whether the beneficial effects of carvedilol are related to microRNAs which have emerged as critical players in cardiovascular pathophysiology via post-transcriptional regulation of protein-coding genes. In vivo, we demonstrated that carvedilol ameliorated impaired cardiac function of infarct rats and restored miR-1<em>3</em><em>3</em> expression. In vitro, carvedilol protected cardiomyocytes from H2O2 induced apoptosis detected by TUNEL staining and MTT assays, and increased miR-1<em>3</em><em>3</em> expression in cardiomyocytes. Overexpression of miR-1<em>3</em><em>3</em>, a recognized anti-apoptotic miRNA, produced similar effects to carvedilol: reduction of reactive oxygen species (ROS) and malondialdehyde (MDA) content and increment of superoxide dismutase (<em>SOD</em>) activity and glutathione peroxidase (GPx) level, so as to protect cardiomyocytes from apoptosis by downregulating caspase-9 and caspase-<em>3</em> expression in the presence of H2O2. Transfection with AMO-1<em>3</em><em>3</em> (antisense inhibitor oligodeoxyribonucleotides) alone abolished the beneficial effects of carvedilol. Caspase-9-specific inhibitor z-LEHD-fmk, caspase-<em>3</em>-specific inhibitor z-DEVD-fmk, caspase-9 siRNA and caspase-<em>3</em> siRNA were used to establish caspase-<em>3</em> as a downstream target of miR-1<em>3</em><em>3</em>. In conclusion, our data indicated that carvedilol protected cardiomyocytes by increasing miR-1<em>3</em><em>3</em> expression and suppressing caspase-9 and subsequent apoptotic pathways.

OBJECTIVE

Resveratrol could induce basal autophagy through the activation of sirtuin. In this study, we investigated the effect of resveratrol on oxidative injury of human umbilical endothelial vein cells (HUVECs) induced by oxidized low-density lipoprotein (ox-LDL) and the role of autophagy in this effect.

METHODS

HUVECs were exposed to 100 mg/L ox-LDL for 24 h to cause oxidative injury. The effect of different concentrations of resveratrol on oxidative damage in HUVECs treated with ox-LDL was evaluated by MTT assay and superoxide dismutase (<em>SOD</em>) activity test. The autophagic level in different groups was measured by the protein expression of microtubule-associated protein 1 light chain <em>3</em> (LC<em>3</em>) and sequestosome 1 (SQSTM1/P62). Autophagosomes were observed under electron microscope and fluorescence microscope (by MDC staining). The expression of silencing information regulator1 (Sirt1) and AMP activated protein kinaseα1 (AMPK) was investigated by Western blot. Autophagy inhibitor <em>3</em>-methyladenine (<em>3</em>-MA) and Sirt1 inhibitor 6-Chloro-2,<em>3</em>,4,9-tetrahydro-1H-Carbazole-1-carboxamide (EX527) were used to confirm the role of autophagy in this effect of resveratrol and the pathway involved.

RESULTS

Resveratrol reversed the decreases in cell viability (72.9 ± 1.7 % of the control group) and <em>SOD</em> activity (14.<em>3</em>7 ± 0.21 U/ml) caused by ox-LDL at 8<em>3</em>.4 ± 1.4 % of the control group and 16.41 ± 0.27 U/ml respectively. This effect accompanied by upregulation of autophagy and increased protein expression of Sirt1 and AMPK phosphorylation on threonine 172 (p-AMPK). Both <em>3</em>-MA and EX527 abolished the protective effect of resveratrol in cell viability, at 80.4 ± 2.7 % and 7<em>3</em>.9 ± 1.1 % of the control group respectively. <em>3</em>-MA inhibited autophagy activation without any change of Sirt1 expression at both the mRNA and protein level. EX527 suppressed the expression of Sirt1 and diminished the upregulation of autophagy. Addition of <em>3</em>-MA or EX527 could not affect the protein level of p-AMPK.

CONCLUSIONS

Resveratrol protected HUVECs from oxidative damage caused by ox-LDL. This effect was mediated by Sirt1-dependent autophagy via the AMPK/ Sirt1 pathway.

BACKGROUND

Large body of evidences accumulated in clinical and epidemiological studies indicate that hearts of diabetic subjects are more sensitive to ischemia reperfusion injury (IRI), which results in a higher rate of mortality at post-operation than that of non-diabetes. However, experimental results are equivocal and point to either increased or decreased susceptibility of the diabetic hearts to IRI, especially at the early stage of the disease. The present study was designed to test the hypothesis that the duration/severity of the indexed ischemia is a major determinant of the vulnerability to myocardial IRI at early stage of diabetes.

METHODS

Four weeks streptozotocin (STZ)-induced diabetic (D) and non-diabetic (C) Sprague-Dawley rats were randomly assigned to receive <em>3</em>0 or 45 min of left anterior descending artery ligation followed by 2 or <em>3</em> hours of reperfusion, respectively. Cardiac function was recorded by using Pressure-Volume (PV) conduction system. Myocardial infarct size was determined with triphenyltetrazolium chloride staining. Plasma Creatine kinase-MB (CK-MB), Lactate dehydrogenase (LDH) release, myocardial nitric oxide(NO) content and nitrotyrosine formation, 15-F(2t)-Isoprostane and plasma superoxide dismutase (<em>SOD</em>) were measured with colorimetric assays. Cardiomyocyte apoptosis was assessed by TUNEL staining. Myocardial TNFα, Caspase-<em>3</em>, STAT<em>3</em>, Akt, and GSK-<em>3</em>β were determined by Western blotting.

RESULTS

Prolongation of ischemia but not reperfusion from <em>3</em>0 min to 45 min significantly increased infarct size in D compared to C rats (P < 0.05), accompanied with significantly increased plasma CK-MB (P < 0.05). Prolongation of the duration of either ischemia or reperfusion significantly increased plasma LDH release and myocardial 15-F(2t)-Isoprostane and reduced plasma <em>SOD</em> activity, with concomitant reduction of myocardial NO and increase of nitrotyrosine formation in D relative to C (P < 0.05). Prolongation of ischemia and reperfusion significantly reduced left ventricular ejection fraction and increased the peak rate of pressure, accompanied with increased end systolic pressure in D relative to C rats (P < 0.05) but reduced phosphorylations of myocardial STAT<em>3</em> at site Ser727 and Akt at site Ser47<em>3</em> as well as GSK-<em>3</em>β at Ser 9 (P < 0.05).

CONCLUSIONS

Diabetic hearts, even at early stage of the disease are more sensitive to IRI, and this increased severity of post-ischemic myocardial injury depends more on the duration of ischemia than that of reperfusion.

Oxidative stress is implicated in the pathogenesis of osteoarthritis. Curcuminoids are natural polyphenols with strong antioxidant capacity and may thus be helpful in the treatment of osteoarthritis. The present randomized double-blind placebo-controlled trial investigated the efficacy of curcuminoids in reducing systemic oxidative burden in patients suffering from knee osteoarthritis. Forty patients with mild-to-moderate primary knee osteoarthritis were given curcuminoid capsules (1500 mg/day in <em>3</em> divided doses; n = 19) or matched placebo capsules (n = 21) for a period of 6 weeks. Curcuminoids were co-administered with piperine (15 mg/day) in order to improve the bioavailability. Serum activities of superoxide dismutase (<em>SOD</em>) and concentrations of reduced glutathione (GSH) and malonedialdehyde (MDA) were determined spectrophotometrically at baseline and at the end of the treatment period in both groups. Serum activities of <em>SOD</em> as well as GSH and MDA concentrations were comparable between the study groups at baseline (p>> 0.05). There was a significant elevation in serum <em>SOD</em> activities (mean change: 2.94 ± <em>3</em>.7<em>3</em> vs. -0.<em>3</em>8 ± 1.<em>3</em><em>3</em>; p < 0.001), a borderline significant elevation in GSH concentrations (mean change: 1.<em>3</em>9 ± 2.78 vs. -0.02 ± 1.62; p = 0.064) and a significant reduction in MDA concentrations (mean change: -5.26 ± 4.46 vs. -2.49 ± <em>3</em>.81; p = 0.044) in the curcuminoids compared with the placebo group. Changes in serum activities of <em>SOD</em> and concentrations of GSH and MDA during the course of trial were significantly correlated. Short-term supplementation with curcuminoids attenuates systemic oxidative stress in patients with osteoarthritis. These antioxidant effects may account for the reported therapeutic effects of curcuminoids in relieving osteoarthritis symptoms.

Antioxidants are one of the key players in tumorigenesis, several natural and synthetic antioxidants were shown to have anticancer effects. In the present investigation the efficacy of silymarin on the antioxidant status of N-nitrosodiethylamine (NDEA) induced hepatocarcinogenesis in Wistar albino male rats were assessed. The animals were divided into five groups. The animals in the groups 1 and <em>3</em> were normal control and silymarin control, respectively. Groups 2, 4 and 5 were administered with 0.01% NDEA in drinking water for 15 weeks to induce hepatocellular carcinoma (HCC). Starting 1 week prior to NDEA administration group 4 animals were treated with silymarin in diet for 16 weeks, 10 weeks after NDEA administration group 5 animals were treated with silymarin and continued till the end of the experiment period (16 weeks). After the experimental period the body weight, relative liver weight, number of nodules, size of nodules, the levels of lipid peroxidation, glutathione (GSH), and the activities of antioxidant enzymes were assessed in both haemolysate and liver tissue. In group 2 hepatocellular carcinoma induced animals there was an increase in the number of nodules, relative liver weight. The levels of lipid peroxides were elevated with subsequent decrease in the body weight, (glutathione) GSH, superoxide dismutase (<em>SOD</em>), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G6PD). In contrast, silymarin + NDEA treated groups 4 and 5 animals showed a significant decrease in the number of nodules with concomitant decrease in the lipid peroxidation status. The levels of GSH and the activities of antioxidant enzymes in both haemolysate and liver were improved when compared with hepatocellular carcinoma induced group 2 animals. The electron microscopy studies were also carried out which supports the chemopreventive action of the silymarin against NDEA administration during liver cancer progression. These findings suggest that silymarin suppresses NDEA induced hepatocarcinogenesis by modulating the antioxidant defense status of the animals.

OBJECTIVE

The protective effects of Ginkgo biloba phytosomes (GBP) on carbon tetrachloride (CCl4)-induced hepatotoxicity and the probable mechanism(s) involved in this protection were investigated in rats.

METHODS

Liver damage was induced in Wistar rats by administering a 1:1 (v/v) mixture of CCl4 and olive oil (1 ml/kg, i.p.) once daily for 7 days. GBP at 25 mg/kg and 50 mg/kg, i.p. and reference drug silymarin (200 mg/kg, p.o.) were administered for 10 days to CCl4-treated rats, this treatment beginning <em>3</em> days prior to the commencement of CCl4 administration. The degree of protection was evaluated by determining the marker enzymes (SGOT, SGPT and SALP), albumin (Alb) and total proteins (TP). Further, the effects of GBP on lipid peroxidation (LPO), glutathione (GSH), superoxide dismutase (<em>SOD</em>), catalase (CAT), glutathione peroxidase (GPX) and glutathione reductase (GR) were estimated in liver homogenates to evaluate antioxidant activity.

RESULTS

GBP (25 and 50 mg/kg) and silymarin elicited significant hepatoprotective activity by decreasing the activities of serum marker enzymes and lipid peroxidation and elevated the levels of GSH, SOD, CAT, GPX, GR, Alb and TP in a dose-dependent manner.

CONCLUSIONS

The present findings indicate that the hepatoprotective effects of GBP against CCl4-induced oxidative damage may be due to its antioxidant and free radical-scavenging activity.

BACKGROUND

Endothelial progenitor cells (EPCs), especially late EPCs, play a critical role in endothelial maintenance and repair, and postnatal vasculogenesis. Advanced glycation end products (AGEs) have been shown to impair EPC functions, such as proliferation, migration and adhesion. However, their role in the regulation of the production of vasoactive substances in late EPCs is less well defined.

METHODS

Passages of <em>3</em>~5 EPCs, namely late EPCs, were cultured with different concentrations (0~500 μg/ml) of AGEs, and the apoptosis, adhesion and migration were subsequently determined. The release of vasoactive substances, such as stromal cell-derived factor-1 (SDF-1), nitric oxide (NO), prostaglandin I2 (PGI2), plasminogen activator inhibitor-1 (PAI-1), tissue plasminogen activator (tPA), and in addition the activity of superoxide dismutase (<em>SOD</em>), were evaluated by ELISA. At the same time, the gene and protein expressions of CXCR4 were assayed by real-time RT-PCR and western-blot.

RESULTS

AGEs promoted late EPC apoptosis. Moreover, AGEs impaired late EPC migration and adhesion in a concentration-dependent manner. Accordingly, the production of SDF-1 was decreased by AGEs. Although the CXCR4 expressions of late EPCs were up-regulated for AGE concentrations of 50, 100 or 200 μg/ml, a marked decrease was observed for the higher concentration of 500 μg/ml. Furthermore, co-culturing with AGEs decreased the levels of NO, t-PA, PGI2, and the activity of SOD but up-regulated the production of PAI-1.

CONCLUSIONS

Our data provide evidence that AGEs play an important role in impairing late EPC functions, which could contribute to the development of vascular diseases in diabetes.

Here we investigated H2O2 production and detoxification in the hematophagous hemiptera, Rhodnius prolixus. Superoxide dismutase (<em>SOD</em>) catalyzes the dismutation of superoxide radical (O2-). This reaction produces hydrogen peroxide, which is scavenged by antioxidant enzymes such as catalase (CAT). <em>SOD</em> and CAT activities were found in all tissues studied, being highest in the midgut. CAT was dose-dependently inhibited in vivo by injections of <em>3</em>-amino-1,2,4-triazole (AT). Insects treated with AT showed a twofold increase in H2O2 levels. Injection of DL-buthionine-[S, R]-sulfoximine (BSO), an inhibitor of glutathione synthesis, also resulted in a fourfold increase in H2O2, together with stimulation of CAT activity. Simultaneous administration of both AT and BSO had a synergistic effect on midgut H2O2 content. Taken all together, our results suggest that CAT and glutathione-dependent mechanisms cooperate to control H2O2 concentration in the midgut cell and prevent hydroxyl radical generation by Fenton reaction in this tissue.

Excessive generation of reactive oxygen species (ROS) has been suggested as a causal factor in various neurodegenerative disorders, such as Parkinson's disease and Alzheimer's disease [Brain Res. 8<em>3</em>0 (1999) 10-15; Biochem. J. <em>3</em>10 (1995) 8<em>3</em>-90; Free Radic. Biol. Med. 27 (1999) 612-616]. The present work examined the role of ROS in the neurotoxicity of methylmercury (MeHg). ROS formation in primary astrocytic cultures of neonatal rat cerebral cortex was monitored by 2',7'-dichlorodihydrofluorescein diacetate (H(2)DCF-DA) fluorescence. MeHg, at 10 and 20 microM caused a significant increase in ROS formation (10 microM, P<0.01; 20 microM, P<0.001). Additional studies established the effectiveness of antioxidants/free radical scavengers in attenuating the MeHg-stimulated ROS formation in the following rank-order: (1) Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a non-thiol containing antioxidant, (2) n-propyl gallate (PG), a free radical scavenger, (<em>3</em>) superoxide dismutase (<em>SOD</em>), an antioxidant enzyme that dismutates superoxide anion radical, (4) alpha-phenyl-tert-butyl nitrone (PBN), a lipophilic hydroxyl radical spin trapping agent. A significant inhibition of MeHg-induced ROS generation was also noted in astrocytes preincubated (<em>3</em> h) with arachidonyl trifluoromethyl ketone (AACOCF(<em>3</em>,) 20 microM, P<0.05), a specific inhibitor of cytosolic phospholipase A(2) (cPLA(2)). Conversely, pretreatment (24 h) with 100 microM buthionine-L-sulfoxamine [BSO, a glutathione (GSH) synthesis inhibitor], significantly increased (P<0.05) ROS formation in MeHg treated astrocytes compared to controls. Combined, these studies invoke ROS as potent mediators of MeHg cytotoxicity and support the hypothesis that excessive ROS generation, at least in part, plays an important role in MeHg-induced neurotoxicity.

In the present study the effect of thyroid hormone (T(<em>3</em>)) on oxidative stress parameters of mitochondria of rat liver is reported. Hypothyroidism is induced in male adult rats by giving 0.05% propylthiouracil (PTU) in drinking water for <em>3</em>0 days and in order to know the effect of thyroid hormone, PTU-treated rats were injected with 20 microg T(<em>3</em>)/100 g body weight/day for <em>3</em> days. The results of the present study indicate that administration of T(<em>3</em>) to hypothyroid (PTU-treated) rats resulted in significant augmentation of oxidative stress parameters such as thiobarbituric acid reactive substances and protein carbonyl content of mitochondria in comparison to its control and euthyroid rats. The hydrogen peroxide content of the mitochondria of liver increased in hypothyroid rats and was brought to a normal level by T(<em>3</em>) treatment. Induction of hypothyroidism by PTU treatment to rats also resulted in the augmentation of total and CN-sensitive superoxide dismutase (<em>SOD</em>) activities of the mitochondria, which was reduced when hypothyroid rats were challenged with T(<em>3</em>). Although CN-resistant <em>SOD</em> activity of the mitochondria remained unaltered in response to hypothyroidism induced by PTU treatment, its activity decreased when hypothyroid rats were injected with T(<em>3</em>). The catalase activity of the mitochondria decreased significantly by PTU treatment and was restored to normal when PTU-treated rats were given T(<em>3</em>). Total, Se-independent and Se-dependent glutathione peroxidase activities of the mitochondria were increased following PTU treatment and reduced when T(<em>3</em>) was administered to PTU-treated rats. The reduced and oxidised glutathione contents of the mitochondria of liver increased significantly in hypothyroid rats and their level was restored to normal when hypothyroid rats were injected with T(<em>3</em>). The results of the present study suggest that the mitochondrial antioxidant defence system is considerably influenced by the thyroid states of the body.

Caloric restriction (CR) extends lifespan through a reduction in oxidative stress, delays the onset of morbidity and prolongs lifespan. We previously reported that long-term CR hastened clinical onset, disease progression and shortened lifespan, while transiently improving motor performance in G9<em>3</em>A mice, a model of amyotrophic lateral sclerosis (ALS) that shows increased free radical production. To investigate the long-term CR-induced pathology in G9<em>3</em>A mice, we assessed the mitochondrial bioenergetic efficiency and oxidative capacity (CS--citrate synthase content and activity, cytochrome c oxidase--COX activity and protein content of COX subunit-I and IV and UCP<em>3</em>-uncoupling protein <em>3</em>), oxidative damage (MDA--malondialdehyde and PC--protein carbonyls), antioxidant enzyme capacity (Mn-<em>SOD</em>, Cu/Zn-<em>SOD</em> and catalase), inflammation (TNF-alpha), stress response (Hsp70) and markers of apoptosis (Bax, Bcl-2, caspase 9, cleaved caspase 9) in their skeletal muscle. At age 40 days, G9<em>3</em>A mice were divided into two groups: Ad libitum (AL; n = 14; 7 females) or CR (n = 1<em>3</em>; 6 females), with a diet equal to 60% of AL. COX/CS enzyme activity was lower in CR vs. AL male quadriceps (<em>3</em>5%), despite a 2.<em>3</em>-fold higher COX-IV/CS protein content. UCP<em>3</em> was higher in CR vs. AL females only. Mn<em>SOD</em> and Cu/Zn-<em>SOD</em> were higher in CR vs. AL mice and CR vs. AL females. MDA was higher (8<em>3</em>%) in CR vs. AL red gastrocnemius. Conversely, PC was lower in CR vs. AL red (62%) and white (<em>3</em>0%) gastrocnemius. TNF-alpha was higher (52%) in CR vs. AL mice and Hsp70 was lower (62%) in CR vs. AL quadriceps. Bax was higher in CR vs. AL mice (41%) and CR vs. AL females (52%). Catalase, Bcl-2 and caspases did not differ. We conclude that CR increases lipid peroxidation, inflammation and apoptosis, while decreasing mitochondrial bioenergetic efficiency, protein oxidation and stress response in G9<em>3</em>A mice.

D-methionine (D-met) protects against cisplatin (CDDP) ototoxicity, but the mechanisms are not well understood. This study investigated D-met protection of cochlear oxidative state as measured by superoxide dismutase (<em>SOD</em>), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR), and malondiadehyde (MDA) levels. The design comprised four groups of five rats each: (1) a saline control group, (2) a CDDP-only-treated group, (<em>3</em>) a CDDP group pretreated with D-met, and (4) a group receiving only D-met. Auditory brainstem response testing (ABR) was performed before and <em>3</em> days after injection. CDDP alone caused marked hearing loss; significantly reduced <em>SOD</em>, CAT, and GR levels; and increased MDA levels, but D-met pretreatment protected against these changes. These studies suggest that D-met protects cochlear antioxidant enzyme levels from CDDP-induced decrements. The excellent correlation of enzyme levels with hearing loss and weight loss suggests that antioxidant enzyme level protection may underlie, at least in part, D-met's otoprotective action.

BACKGROUND

Chronic exposure to stress alters the normal body homeostasis and, hence, leads to the development of various human pathologies, which might involve alterations in the antioxidant defense system. We studied the effect of chronic cold exposure on oxidative stress and antioxidant defense system in various rat tissues.

METHODS

Male albino rats (Wistar strain), 2-<em>3</em> months old, were exposed to <em>3</em> weeks of cold treatment. Antioxidant enzymes, superoxide dismutase (<em>SOD</em>), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione S-transferase (GST) were measured in addition to the antioxidants, ascorbic acid (AsA) and glutathione (GSH), and the prooxidants, lipid peroxides (LPO) and xanthine oxidase (XOD), in brain, heart, kidney, liver and small intestine using standard protocols.

RESULTS

Chronic cold exposure resulted in a significant increase in LPO in all the tissues studied while XOD was increased in the brain and intestine. Total SOD activity was significantly decreased in all the tissues, whereas CAT activity was significantly increased in the kidney and decreased in heart, liver and intestine in the animals exposed to cold. GPx activity was increased only in the brain and intestine of stressed rats. Chronic cold exposure resulted in significant decrease in GR activity in heart, liver and intestine. GST activity was increased (except heart) and GSH was significantly decreased in all the tissues in treated rats. AsA was increased in kidney and intestine but decreased in heart of stressed animals.

CONCLUSIONS

The observed changes in the antioxidant defense system are tissue specific, but it is evident that chronic exposure to cold leads to oxidative stress by displacing the prooxidant-antioxidant balance of this defense system by increasing the prooxidants while depleting the antioxidant capacities.

We showed previously that the dietary combination of fish oil, rich in (n-<em>3</em>) fatty acids, and the fermentable fiber pectin enhances colonocyte apoptosis in a rat model of experimentally induced colon cancer. In this study, we propose that the mechanism by which this dietary combination heightens apoptosis is via modulation of the colonocyte redox environment. Male Sprague-Dawley rats (n = 60) were fed 1 of 2 fats (corn oil or fish oil) and 1 of 2 fibers (cellulose or pectin) for 2 wk before determination of reactive oxygen species (ROS), oxidative DNA damage, antioxidant enzyme activity [superoxide dismutase (<em>SOD</em>), catalase (CAT), glutathione peroxidase (GPx)] and apoptosis in isolated colonocytes. Fish oil enhanced ROS, whereas the combination of fish oil and pectin suppressed <em>SOD</em> and CAT and enhanced the <em>SOD</em>/CAT ratio compared with a corn oil and cellulose diet. Despite this modulation to a seemingly prooxidant environment, oxidative DNA damage was inversely related to ROS in the fish oil and pectin diet, and apoptosis was enhanced relative to other diets. Furthermore, apoptosis increased exponentially as ROS increased. These results suggest that the enhancement of apoptosis associated with fish oil and pectin feeding may be due to a modulation of the redox environment that promotes ROS-mediated apoptosis.

Proteoglycans and plasma proteins bound to the endothelial cell glycocalyx are essential for vascular function, but at the same time, they lower capillary tube hematocrit by reducing capillary volume available to flowing blood. Because oxidized low-density lipoproteins (oxLDL) reduce the effective thickness of the glycocalyx (Vink H, Constantinescu AA, and Spaan JAE. Circulation 101: 1500-1502, 2000), we designed the present study to determine whether this is caused by pathological degradation of glycocalyx constituents or increased glycocalyx deformation by elevated shear forces of flowing blood. Capillaries from the right cremaster muscle of 24 hamsters were examined by using intravital microscopy after systemic administration of normal LDL (n = 4), moderate oxLDL (6-h oxidation with CuSO(4), n = 7), severe oxLDL (18-h oxidation, n = 5), and moderate oxLDL plus superoxide dismutase (<em>SOD</em>) and catalase (n = 8). Capillary tube hematocrit increased from 0.16 +/- 0.0<em>3</em> to 0.<em>3</em>7 +/- 0.05 and from 0.15 +/- 0.01 to 0.<em>3</em>1 +/- 0.0<em>3</em> after moderate oxLDL and severe oxLDL, respectively. These changes were paralleled by increases in red blood cell flux from 8.7 +/- 1.9 to 1<em>3</em>.8 +/- <em>3</em> and from 10.7 +/- 2.1 to 16.<em>3</em> +/- <em>3</em>.2 cells/s after moderate oxLDL and severe oxLDL, respectively, in the absence of changes in anatomic capillary diameter. Red blood cell velocity, as a measure for the shear forces on the glycocalyx, was not affected by oxLDL, whereas tissue pretreatment with <em>SOD</em> and catalase completely abolished the effects of oxLDL on glycocalyx thickness, capillary hematocrit, and red blood cell flux. We conclude that elevation of capillary tube hematocrit by oxLDL reflects degradation of the endothelial glycocalyx by oxygen-derived free radicals.